Vitamin D3 analogues

ABSTRACT

A vitamin D 3  analogue which includes at least one hydroxyalkyl substituent on the A-ring, preferably the 1-position thereof.

The present invention relates to novel biologically active vitamin D₃analogues which include at least one hydroxyalkyl substituent on theA-ring.

The research disclosed herein was supported by a grant from The NationalInstitutes of Health.

BACKGROUND OF THE INVENTION

Vitamin D₃ analogues have been recognized as having important biologicalactivities. It is known, for example, that vitamin D₃ analogues can beused to control calcium and phosphate metabolism.

It is also known that such analogues are useful for inducing celldifferentiation and for inhibiting undesired cell proliferation. Forexample, it is well recognized that during normal metabolism vitamin D₃produces 1α,25-dihydroxyvitamin D₃ (calcitriol) which is a potentregulator of cell differentiation and proliferation as well asintestinal calcium and phosphorus absorption and bone calciummobilization. Calcitriol is also known to affect the immune system andthis compound, as well as a variety of synthetic vitamin D₃ derivativeshave been used in practical, clinical chemotherapy of such diverse humanillnesses as osteoporosis, cancer, immunodeficiency syndromes and skindisorders such as dermatitis and psoriasis. However, major researchefforts are underway in an effort to prepare vitamin D₃ analogues asdrugs in which calcitropic activity is effectively separated from cellgrowth regulation.

Numerous references can be cited as showing prior work with respect tovitamin D₃ analogues, calcitriol or the like. See, for example: VitaminD. Chemical, Biochemical, and Clinical Update, Proceedings of the SixthWorkshop on Vitamin D, Merano, Italy, March 1985; Norman, A. W.,Schaefer, K., Grigoleit, H. G., Herrath, D. V. Eds.; W. de Gruyter; NewYork, 1985; Brommage, R., DeLucca, H. F., Endocrine Rev., 1985, 6, 491;Dickson, I., Nature, 1987, 325, 18; Cancela, L., Theofon, G., Norman, A.W., in Hormones and Their Actions, Part I; Cooke, B. A., King, R. J. B.,Van der Molen, H. J. Eds.; Elaevier, Holland, 1988; Tsoukas, D. C.,Provvedini, D. M., Manolagas, S. C., Science, (Washington, D.C.) 1984,224, 1438; Provvedini, D. M., Tsoukas, C. D., Deftoe, L. J., Manolagas,S. C., Science, (Washington, D.C.) 1983, 221, 1181; Vitamin D. ChemicalBiochemical, and Clinical Endocrinology of Calcium Metabolism,Proceedings of the Fifth Workshop on Vitamin D, Williamsburg, Va.,February 1982, Norman, A. W., Schaefer, K., Herrath, D. V., Grigoleit,H. G., Eds., W. de Gruyter, New York, 1982, pp. 901-940; Calverley, M.J. in Vitamin D: Molecular, Cellular, and Clinical Endocrinology,Norman, A. W., Ed., de Gruyter; Berlin, 1988, p. 51; Calverley, M. J.,Tetrahedron. 1987, 43, 4609.

Calcitriol may be structurally represented as follows: ##STR1##

The upper and lower ring portions of calcitriol may be called, for easeof reference, the C/D-ring and A-ring, respectively.

Many analogues of calcitriol have been synthesized and evaluated. Amongthese, all the leading candidates include the 1α-hydroxyl A-ringsubstituent characteristic of calcitriol, i.e. they differ in the sidechain attached to the D-ring of the steroid framework. Some calcitriolanalogues lacking the 1α-hydroxyl group have also been prepared, e.g.the 1β-hydroxyl, 1α-fluoro and the 1-unfunctionalized (i.e.25-hydroxyvitamin D₃). However, these have been found to be much lessbiologically active than calcitriol and other synthesized 1α-hydroxyanalogues. Accordingly, it appears to be axiomatic among workers in thefield that the 1α-hydroxy group is essential for desirable biologicalactivity. See, for example, Biochem Biophys Res. Commun., 97:1031(1980); Chem. Pharm. Bull., 32:3525 (1984) and Bull. Soc. Chim. France,II:98 (1985).

SUMMARY OF THE INVENTION

The present invention is based on the unexpected finding that the A-ringportion of vitamin D₃ analogues can be modified without negativelyaffecting the biological activity of the resulting compounds. In itsbroadest aspects, the invention provides vitamin D₃ analogues whichinclude at least one hydroxyalkyl substituent on the ring-A. It iscontemplated that this hydroxyalkyl substituent may be placed on the1,2,3- and/or 4-positions of the A-ring. However, the preferredembodiment of the invention is the vitamin D₃ analogue wherein the1α-hydroxy group is replaced by a hydroxyalkyl group of, for example,1-6 carbon atoms.

Structurally, the preferred D₃ analogues of the invention may be shownas follows: ##STR2## including the stereoisomers thereof, wherein R is-R³ OH, R³ being straight or branched alkyl of 1 to 6 carbons; R¹ ishydrogen and R² represents the substituents completing a vitamin D₃analogue.

However, also contemplated are the corresponding analogues which includeone or more hydroxyalkyl substituents in the 2,3- and/or 4-position inlieu of, or in addition to, the hydroxyalkyl in the 1-position of thering-A.

It will be appreciated that the D-ring may include the conventional D₃substitution or any other known modification thereof. See, for example,the variations shown in Cancer Research. 50:6857-6864 (Nov. 1, 1990),the contents of which are incorporated herein by reference.

The preferred compound according to the invention is1-hydroxymethyl-25-hydroxyvitamin D₃ represented by the formula:##STR3## or the formula:

However, as noted, the invention is not to be viewed as limited to thesecompounds as other analogues involving the attachment of one or moreadditional hydroxyalkyl groups on the ring-A, with various othermodifications as substituents in the ring-D, are contemplated.

DETAILED DESCRIPTION OF THE INVENTION

Preferred procedures for preparing the 1-hydroxyalkyl analogues of theinvention are shown hereinafter although it will be appreciated thatother procedures or modifications thereof can be used and will beevident to those in the art. Thus, the preparation of the twodiastereomeric forms of 1-hydroxymethyl-25-hydroxyvitamin D₃, isillustrated, but not limited, by the following reaction Schemes I-III inconjunction with the examples which follow: ##STR4##

The reaction scheme illustrated in Scheme III hereinafter utilizemethodology described earlier (J. Oro. Chem., 56:4339 (1991); ibid57:000 (1992); Tetrahedron Lett., 32:5295 (1991); J. Org. Chem., 55:3967(1990) and Accts. Chem. Res., 20:72 (1987)) to prepare ring-A phosphineoxide 11 for Horner-Wittig coupling with C,D-ring ketone 12 in aconvergent approach to the vitamin D₃ family that was pioneered byLythgoe et al (J. Chem. Soc., Perkin I, 2608 (1977)).

The preparation process begins, as shown in Scheme I, using ambiphilic(chameleon-like) 3-bromo-2-pyrone (4) to undergo regiospecific, andstereoselective Diels-Alder cycloaddition with acrolein undersufficiently mild thermal conditions (70°-90° C.) to allow isolation ongram scale of the desired, unsaturated, bridged, bicyclic lactoneadduct. Because this bicyclic aldehyde was unstable to chromatography,it was immediately reduced and then O-silylated to givechromatographically stable, crystalline, bicyclic, primary alcoholderivative 5 in 46% overall yield. Reductive cleavage of the bridgeheadcarbon-bromine bond was achieved in high yield under neutral radicalconditions using tributyltinhydride and azobisisobutyronitrile (AIBN).The halogen-free bicyclic lactone product is the synthetic equivalent ofthe product derived from 2-pyrone itself cycloadding to acrolein, aDiels-Alder reaction that requires high pressures and that cannot beaccomplished simply by heating because of loss of CO₂ from the lactonebridge. Basic methanolysis of the lactone bridge and in situ conjugationof the carbon-carbon double bond gives the conjugated cyclohexene esteralcohol 6. Resolution of this alcohol 6 is achieved via formation andseparation by preparative HPLC and preparative tlc of diastereomericesters 7a and 7b, derived from enantiomerically pureα-methoxyphenylacetic acid. Analytical HPLC indicated purifieddiastereomer 7a to have a diastereomeric excess (d.e.) of 98.8% and 7bof 96.5%. Methanolysis of diastereomeric esters 7a and 7b separatelygave back the original alcohol 6 as a pair of enantiomers, 6a and 6b;each enantiomer was carried on separately.

The absolute stereochemistry of enantiomer 6b (and therefore also 6a)has been assigned by chemical correlation with a closely relatedcompound of established absolute configuration (J. Chem. Soc., (C), 2352(1971)), as outlined in Scheme II. ##STR5##

Referring back to Scheme I, O-silylation of alcohols 6 gave bis-silylethers 8, and then reduction of the conjugated methyl esterfunctionality produced allylic alcohols 9. [3,3]-Sigmatropicrearrangement using sulfinyl orthoester allowed efficient, one-flask,regiospecific formation of 2-carbon-extended conjugated dienoate esters10 (J. Org. Chem., 56:6981 (1991)). This mixture of geometric isomerswas photochemically isomerized into the desired Z-10. Based onliterature precedent (J. Org. Chem., 51:3098 (1986)), dienoate esters 10were reduced, chlorinated, converted into the corresponding phosphines,and finally oxidized to give ring-A phosphine oxides 11 as twoenantiomers (11a and 11b) having almost equal but opposite specificrotations of approximately 54°.

Lythgoe-type coupling (J. Chem. Soc., Perkin I, 2608 (1977)) of 60-100mg of ring-A phosphine oxides 11a and 11b with enantiomerically purering-C,D chiron 12 was followed immediately by fluoride-promoteddesilylation to form (-)-1α-hydroxymethyl-25-hydroxyvitamin D₃ [(-)-2]and (+)-1β-hydroxymethyl-3α,25-hydroxy analogue (+)-3 in good yields(Scheme III). Two aspects of this coupling should be noted inparticular. First, a systematic study of bases used to deprotonatephosphine oxides like 11 (e.g., MeLi, MeLi.TMEDA, n-BuLi, PhLi, LDA)showed PhLi to be best as determined by the yield of the coupled trieneproduct. Second, the scale of the coupling reaction was critical to itssuccess. Thus, while coupling using 60-100 mg of ring-A phosphine oxideproceeded routinely in good yields, coupling on 10-20 mg scale proceededpoorly even if such special precautions were taken such as scrupulousdrying of the gaseous nitrogen or argon gas used as the atmosphere abovethe reaction mixture, scrupulous drying of solvents and reagents, use ofmolecular sieves, and azeotroping off any adventitious water by addingand removing benzene from the A and the C,D-ring units repeatedly.##STR6##

While both 1-hydroxymethyl-25-hydroxyvitamin D₃ diastereomers (-)-2 and(+)-3 demonstrate useful biological activity, it is surprising to findthat there are considerable physical difficulties between thesediastereomers. For example, whereas 1α-hydroxymethyl diastereomer (-)-2is easily crystallized, 1β-hydroxymethyl diastereomer (+)-3 is verydifficult to crystallize. This difference in crystallinity offers asignificant advantage since a mixture of diastereomers (-)-2 and (+)-3,produced from racemic ring-A phosphine oxide 11 and enantiomericallypure ring-C,D chiron 12, could be induced to yield crystals of onlydiastereomer (-)-2. Also, 1α-hydroxymethyl diastereomer (-)-2demonstrates unexpectedly poor solubility in such organic solvents asmethylene chloride, chloroform and methanol. Nevertheless, bothhydroxymethyl diastereomers (-)-2 and (+)-3 have extremely similar UVand high field ¹ H and ¹³ C NMR spectra as well as extremely similarchromatographic properties.

EXAMPLE 1 Bromobicyclic Lactone 5

A 25 mL hydrolysis tube was charged with 1.43 g (8.2 mmol, 1.0 eq.) of3-bromo-2-pyrone 4, 3.69 g (65.7 mmol, 8.0 eq.) of acrolein, 23.0 mg ofbarium carbonate and 10 mL of methylene chloride. This was sealed undernitrogen and warmed to 70°-90° C. for 91 hours with constant stirring.Examination of an aliquot of the reaction mixture by 400 MHz ¹ H NMRindicated that complete formation of a single bicycloadduct hadoccurred. A stream of nitrogen was then blown over the reaction mixtureso as to remove the acrolein. After holding this under high vacuum, thecrude product was diluted with methylene chloride/diethyl ether (ca.1:1) and passed through a plug of celite. The solvent was evaporated togive 3.32 g of a yellow oil which was dissolved in 50 mL ethanol and 20mL of diglyme and cooled to -78° C. (dry ice/acetone) under argon. Tothis, a solution of 476 mg (21.6 mmol, 1.5 eq.) of NaBH₄ in 8 mL ofethanol was added. After stirring for 30 minutes, the mixture wasdiluted with methylene chloride and then 4 mL of saturated aqueousammonium chloride was added. After warming to room temperature, thismixture was dried over anhydrous magnesium sulfate, filtered through aplug of celite, and purified by column chromatography (silica gel, 20%to 50% ethyl acetate/hexane) to afford 1.42 g of a yellow oil which wasimmediately dissolved in 20 mL of anhydrous methylene chloride underargon and cooled to 0° C. To this 0.75 mL (6.4 mmol, 1.05 eq.) of2,6-lutidine was added followed by the addition of 1.5 mL (6.5 mmol,1.07 eq.) of tert-butyldimethylsilyl trifluoromethanesulfonate. This wasstirred for 30 minutes, warmed to room temperature, diluted withmethylene chloride, washed with water, the organic portion dried overmagnesium sulfate, and the solvent evaporated. Purification by silicagel column chromatography (10 to 20% ethyl acetate/hexane) afforded 1.32g (3.8 mmol, 46%) of the silyloxy bromo bicycloadduct 5 as a white solid(Rf=0.7, 50% ethyl acetate/hexane), mp 100.5°-102° C. ¹ H NMR (CDCl₃) δ6.37-6.40 (m, 1H), 6.33 (dd, 8, 5 Hz, 1H), 5.18-5.22 (m, 1H), 3.96 (dd,J=10.1, 3.5 Hz, 1H), 3.65 (dd, J=10.1, 7.1 Hz, 1H), 2.43-2.49 (m, 1H),2.31-2.37 (m, 1H), 1.91 (ddd, J=13.2, 3.9, 1.3 Hz, 1H), 0.86 (s, 9H),0.05 (s, 3H), 0.04 (s, 3H); ¹³ C NMR (CDCl₃) δ 169.0, 136.4, 130.4,73.5, 64.3, 62.1, 41.1, 31.2, 25.7 (3C), 18.1, -5.4, -5.5; FT-IR (CHCl₃)1763 cm⁻¹ ; HRMS, m/z (M⁺ -t-Bu) calcd for C₁₄ H₂₃ O₃ SiBr 288.9896,found 288.9901.

EXAMPLE 2 Hydroxy α, β-Unsaturated Ester 6 (from 5)

To a 25 mL flame-dried round-bottomed flask 179.6 mg (0.52 mmol, 1.0eq.) of silyloxy bromo bicycloadduct 3, and a total of 0.20 mL oftri-n-butyltin hydride, 15 mg of azobisisobutyronitrile (AIBN), and 4.0mL of anhydrous benzene was added and refluxed (placed in a preheatedoil bath) for a total of 75 minutes. This was cooled to room temperatureand then diluted with wet ether. A few drops of1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) were added and the mixturestirred for 5 minutes at which time the white precipitate was removed byfiltration through a plug of silica gel with ether. The solvent wasevaporated and the resulting oil placed in a 50 mL flame-driedround-bottomed flask under argon. The oil was dissolved in 3 mL ofanhydrous tetrahydrofuran (THF) and cooled to -45° C. To this, 0.6 mL ofa freshly prepared sodium methoxide solution (20 mg of sodium in 4.0 mLof anhydrous methanol) was added and stirred at -45' C. for 2.5 hoursand then at 25° C. for 1 hour. The reaction mixture was diluted withmethylene chloride, quenched with saturated aqueous ammonium chloride,dried over anhydrous magnesium sulfate, filtered, and the solventevaporated. Purification by silica gel chromatography afforded 119.2 mg(0.40 mmol, 77%) of hydroxy ester 6 as a colorless oil (Rf=0.2, 25%ethyl acetate/hexane). ¹ H NMR (CDCl₃ δ 6.94 (ddd, J=5, 3, 1 HZ, 1H),4.20-4.12 (m, 1H), 3.72 (s, 3H), 3.74-3.71 (m, 1H), 3.50 (dd, J=10.0,8.0 Hz, 1H), 2.90 (bs, 1H), 2.60 (dtdd, j=19.2, 6, 1.6, 1 Hz, 1H), 2.23(dddd, J=12.4, 4, 2.8, 1.6 Hz, 1H), 2.09 (dddd, J=19.2, 8.8, 3.0, 2.0Hz, 1H), 1.65 (bs, 1-OH, this signal disappears upon D₂ O quench), 1.57(ddd, J=12.4, 11.2, 6 Hz, 1H), 0.87 (s, 9H), 0.03 (s, 3H), 0.01 (s, 3H);¹³ C NMR (CD₂ Cl₂) δ 167.4, 139.9, 130.5, 65.1, 63.6, 51.8, 38.1, 35.6,33.8, 26.1 (3C), 18.5, -5.3, -5.4; FT-IR (thin film) 3412, 1716 cm⁻¹ ;HRMS, m/z (M⁺ -t-Bu) calcd for C₁₅ H₂₈ O₄ Si 243.1053, found 243.1059.

EXAMPLE 3 Hydroxy α, β-Unsaturated Ester 6 (from 7)

A round-bottomed flask was charged with 0.632 g (1.41 mmol) of thediester 7b which was dissolved in 10 mL of tetrahydrofuran and 10 mL ofmethanol and then cooled to 0° C. To this, 0.20 mL of a freshly preparedsodium methoxide stock solution (32.1 mg of sodium in 5.0 mL ofmethanol) was added and rapidly stirred for 1 hour and then warmed toroom temperature. Rapid stirring was maintained and the progress of thereaction was monitored by TLC. Periodic addition of sodium methoxidestock solution was made until the reaction was complete (ca. 8 hours).Most of the solvent was evaporated and the mixture was diluted withdiethyl ether and passed through a two-inch plug of silica gel.Purification by silica gel column chromatography (25% to 75% ethylacetate/hexane) gave 0.386 g (1.28 mmol, 91%) of the hydroxy ester(+)-6a as a colorless oil:

[α]_(D) ²³° C. +59.7° (c=0.082, CH₂ Cl₂, d.e. 98.8%

The same procedure was used for the conversion of 0.900 g (2.01 mmol) ofthe diester 7b into 0.548 g (1.82 mmol, 91%) of the hydroxy ester (-)-6bas a colorless oil:

[α]_(D) ²³° C -59.4° (c=0.085, CH₂ Cl₂, d.e. 98.8%

EXAMPLE 4 α-Methoxyphenylacetic Esters 7a and 7b

To flame-dried 250 mL round-bottomed flask 3.11 g (10.4 mmol of hydroxyester 6, 2.06 g (12.4 mmol, 1.2 eq) of (R)-(-)-α-methoxyphenyl aceticacid, 2.45 g (11.9 mmol, 1.15 eq) of 1,3-dicyclohexylcarbodiimide, and0.15 g (1.2 mmol, 0.1 eq) of 4-dimethylaminopyridine were dissolved in150 mL of anhydrous Et₂ O under argon. This reaction mixture was stirredat room temperature for 12 h. The white precipitate was then removed byfiltration, the organic layer was washed twice with water, dried overMgSO₄, and the solvent removed by rotary evaporation to leave a verylight yellow oil. All impurities were removed from the diastereomericester 7a and 7b by silica gel column chromatography (0-20%EtOAc/hexane). The diastereomers were then separated by preparativenormal phase HPLC (4.5% EtOAc/hexane, 30 mL/min) and by preparativethick layer chromatography (PTLC, multiple elutions with 15%EtOAc/hexane, 1500μ plates). On a preparative scale, the diastereomersoverlapped on both HPLC and PTLC; therefore, fractions were cut andrepurified by numerous injections (ca. 8) and applications,respectively. The diastereomeric excess (d.e.) of fractions was deducedby analytical normal phase HPLC (7a: R.sub.τ =13.4; 7b: R.sub.τ =15.1,1.0 mL/min, 10% EtOAc/hexane). A 1.09 g (2.43 mmol, 23%) sample of 7a(d.e. 98.5%) and a 0.90 g (2.01 mmol, 19%) sample of 7b (d.e. 96.5%)were obtained. A 1.22 g (2.72 mmol, 26%) mixture of 7a and 7b was notadequately separated so as to be used in the subsequent synthetictransformations. 7a: ¹ H NMR (CDCl₃) δ 7.44-7.32 (m, 5H), 6.80 (ddd,J=4.7, 3.35, 1.1 Hz, 1H), 5.34-5.24 (m, 1H), 4.75 (s, 1H), 3.72 (s, 3H),3.69 (d, J=3.4 Hz, 1H), 3.57 (dd, J=10, 7.2 Hz, 1H), 3.41 (s, 3H), 2.90(bs, 1H), 2.57-2.51 (m, 1H), 2.20-2.15 (m, 1H), 1.95 (dddd, J=19.1, 8.1,3.35, 1.9 HZ, 1H), 1.72 (ddd, J=12.8, 11.2, 6.0 Hz, 1H), 0.85 (s, 9H),0.02 (s, 3H), 0.01 (s, 3H); ¹³ C NMR (CDCl₃) δ 169.9, 166.5, 137.9,136.1, 130.0, 128.4, 128.3 (2C), 126.9 (2C), 82.4, 67.6, 64.3, 57.1,51.3, 36.7, 30.9, 29.7, 25.7 (3C), 18.0, -5.7, -5.8; FT-IR (thin film)1749, 1716 cm⁻¹ ; HRMS, m/z (M+-t-Bu) calcd for C₂₄ H₃₆ O₆ Si 391.1577,found 391.1580. 7b: ¹ H NMR (CDCl₃) δ 7.43-7.31 (m, 5H), 6.88 (ddd, J=4.75, 3.3 1 Hz, 1H), 5.29-5.21 (m, 1H), 4.73 (s, 1H), 3.71 (s, 3H),3.64 (dd, J=9.9, 3.5 Hz, 1H), 3.52 (dd, J=9.9, 7.1 Hz, 1H), 3.40 (s,3H), 2.77 (bs, 1H), 2.67 (dddd, J=19, 6, ≈4.75, 1 Hz, 1H), 2.16 (ddd,J=19, 8, 3.3, 2 Hz, 1H), 2.00 (m, 1H), 1.59 (12.8, 11.0, 6, 1H), 0.81(s, 9H), -0.03 (s, 3H), -0.07 (s, 3H); ¹³ C NMR (CDCl₃) δ 170.1, 166.7,138.1, 136.2, 130.3, 128.6, 128.5 (2C), 127.0 (2C), 82.5, 67.8, 64.3,57.2, 51.5, 36.7, 31.4, 29.6, 25.7 (3C), 18.1, -5.6, -5.7; FT-IR (thinfilm) 1749, 1716 cm⁻¹ ; HRMS, m/z (M⁺ -t-Bu) calcd for C₂₄ H₃₆ O₆ Si391.1577, found 391.1576.

EXAMPLE 5 Bis Silyloxy α,β-Unsaturated Ester 8

In a 50 mL flame-dried round bottomed flask 202.5 mg (0.67 mmol, 1.0eq.) of hydroxy ester 6 was dissolved in 15 mL of anhydrous methylenechloride under argon. To this 0.100 mL (0.84 mmol, 1.25 eq.) of2,6-lutidine was added and stirred for 3 minutes followed by theaddition of 0.195 mL (0.84 mmol, 1.25 eq.) of tert-butyldimethylsilyltrifluoromethanesulfonate. After 30 minutes, the solvent was evaporatedand purification by silica gel column chromatography (5 to 10% ethylacetate/hexane) gave 240.4 (0.58 mmol, 86%) of the silyloxy ester 8 as acolorless oil (Rf=0.6, 10% ethyl acetate/hexane). ¹ H NMR (CDCl₃) δ 6.92(ddd, J=5.2, 2.8, 1 Hz, 1H), 4.15 (m, 1H), 3.72-3.69 (m, 1H), 3.71 (s,3H), 3.52 (dd, J=9, 8 Hz, 1 H), 2.76 (bs, 1H), 2.47 (dtd, J=19.2, ca.5.2, 1 Hz, 1H), 2.17-2.12 (m, 1H), 2.13-2.05 (dddd, J=19.2, 9, 2.8, 2.0Hz, 1H), 1.58-1.51 (ddd, J=12.8, 11.2, 2.0, Hz, 1H), 0.88 (s, 9H), 0.87(s, 9H), 0.07 (s, 3H), 0.06 (s, 3H), 0.02 (s, 3H), 0.01 (s, 3H); ¹³ CNMR (CD₂ Cl₂) δ 167.4, 140.3, 130.4, 65.3, 64.6, 51.7, 38.4, 36.5, 34.6,26.1 (6C), 18.6, 18.5, -4.4 to -5.3 (4C); FT-IR (thin film) 1716 cm⁻¹ ;HRMS, m/z (M⁺ -t-Bu) calcd for C₂₁ H₄₂ O₄ Si₂ 357.1917, found 357.1922.

(-) -8 from (-)-6b: [α]_(D) ²³° C. -46.7° (c=0.094, CH₂ Cl₂, d.e. 96.5%)

(+) -8 from (+)-6a: [α]_(D) ²³° C. -47.1° (c=0.100, CH₂ Cl₂, d.e. 98.8%)

EXAMPLE 6 Dienoates E-10 and Z-10

A flame-dried 50 mL round-bottomed flask was charged with 240.4 mg (0.58mmol, 1.0 eq.) of the silyloxy ester 8, dissolved in 4.0 mL of anhydroustoluene, and cooled to -78° C. under argon. To this 1.3 mL (1.2 mmol,2.2 eq.) of diisobutylaluminum hydride DIBAL-H (1.0M in hexane) wasadded and stirred at -78° C. for 30 minutes and then at 25° C. for 90minutes. This was quenched with 5 drops of 2N sodium potassium tartrate,1.5 mL of water, and diluted with methylene chloride. This wasseparated, the organic portion dried over anhydrous magnesium sulfate.Purification by silica gel column chromatography (10 to 25% ethylacetate/hexane) gave 194.2 mg (0.050 mmol, 87%) of the allylic alcohol 9as a colorless oil (Rf=0.5, 25% ethyl acetate/hexane) which wasimmediately used in the preparation of E-10 and Z-10. A 25 mL hydrolysistube was charged with 184.7 mg (0.48 mmol, 1.0 eq.) of the allylicalcohol 6, a total of 427 mg (1.5 mmol, 3.1 eq.) of1-phenylsulfinyl-2,2,2-triethoxyethane, 3 mg of 2,4,6-trimethylbenzoicacid, and 9 mL of anhydrous methylene chloride. This was sealed undernitrogen and warmed to 135°-145° C. for a total of 12.5 hours. Aftercooling the reaction mixture, the solvent was evaporated andpurification by PTLC (3×1000μ, 3% ethyl acetate/hexane) gave 141.6 mg(0.31 mmol, 65%) of E-10 and 19.9 mg (0.04 mmol, 9%) of Z-10 as oils.Shorter reaction times lead to increased Z/E ratios.

E-10: ⁻¹ H NMR (CDCl₃) δ 5.84 (t, J=1.4 Hz, 1H), 5.11 (s, 1H), 4.81 (t,J=1.4 Hz, 1H); ¹³ C NMR (CDCl₃) δ 1.664, 158.0, 149.6, 115.4, 111.4,66.7, 65.2, 59.6, 42.2, 38.4, 36.5, 25.8 (3C), 25.7 (3C), 18.1, 18.0,14.3, -4.89, -4.94, -5.48, -5.53; FT-IR (thin film) 1716 cm⁻¹ ; HRMS,m/z (M⁺ -t-Bu) calcd for C₂₄ H₄₆ O₄ Si₂ 397.2230, found 397.2235.

Z-10: ¹ H NMR (CDCl₃) δ 5.58 (t, 1 Hz, 1H), 4.96-4.93 (m, 2H), 4.15-4.04(m, 3H), 3.71 (dd, J=10, 5.0 (Hz, 1H), 3.52 (t, 10 Hz, 1H), 2.75-2.68(m, 1H), 2.44 (ddt, 12.4, 4.0, 1 Hz, 1H), 2.26 (dddd, 12.4, 8.0, 1.6 Hz,1H), 2.03 (dddd, J=13, 5.6, 4.0, 1.6 Hz, 1H), 1.7 (ddd, 13, 4, 1 Hz,1H), 1.23 (t, 7.2 Hz, 3H), 0.089 (s, 9H), 0.087 (s, 9H), 0.06 (s, 6H),0.043 (s, 3H), 0.040 (s, 3H); ¹³ C NMR (CDCl₃) δ 166.3, 154.2, 145.6,116.4, 112.3, 67.5, 64.2, 60.0, 47.2, 44.0, 36.9, 25.84 (3C), 25.75(3C), 18.2, 18.0, 14.0, -4.73, -4.80, -5.42, -5.50; FT-IR (CDCl₃) 1718cm⁻¹ ; HRMS, m/z (M⁺ -t-Bu) calcd for C₂₄ H₄₆ O₄ Si₂ 397.2230, found397.2231.

(-) -E-10 from (+)-8: [α]_(D) ²³° C. -38.0° (c=0.094, CHCl₃, d.e. 98.5%)

(+) -E-10 from (-)-8: [α]_(D) ²³° C. -37.2° (c=0.051, CHCl₃, d.e. 96.5%)

EXAMPLE 7 Photoisomerization to dienoate Z-10

A borosilicate test tube was charged with 141.1 mg (0.31 mmol) ofdienoate E-10, 9.3 mg of 9-fluorenone, and 9.0 mL of tert-butyl methylether. The tube was sealed with a rubber septum, placed in a solution of2M sodium orthovanadate and irradiated with a medium pressure mercuryarc lamp for 16 hours. This was purified by PTLC (1×1000μ, 1×1500μ, 3%ethyl acetate/hexane) to give 132.3 mg of an inseparable mixture of Z-10and 9-fluorenone [therefore, the yield of Z-10 would be 123.0 mg (0.27mmol, 87%); that is, 132.3 mg of starting material minus 9.3 mg offluorenone].

EXAMPLE 8 Phosphine oxide 11

A flame-dried round-bottomed flask was charged with 123.0 mg (0.27 mmol,1.0 eq. containing 9.3 mg of 9-fluorenone) of Z-10 and 1.5 mL ofanhydrous toluene under argon and then cooled to 0° C. To this 0.60 mL(0.60 mmol, 2.2 eq.) of diisobutylaluminum hydride DIBAL-H (1M inhexane) was added and stirred at 0° C. for 35 minutes and then warmed to25° C. An additional 0.06 ml (0.06 mmol, 0.2 eq.) of DIBAL-H was addedand stirred for 2 hours. The reaction mixture was quenched with 0.5 mLof 2N sodium potassium tartrate, diluted with methylene chloride,separated, and the organic portion dried over anhydrous magnesiumsulfate. Purification by PTLC (2×1000μ), (2 elutions) 10% ethylacetate/hexane and then 15% ethyl acetate/hexane gave 56.8 mg (0.14mmol, 51%) of the allylic alcohol as an oil. A flame-dried 25 mLround-bottomed flask was charged with 90 mg (0.67 mmol, 4.8 eq.) ofN-chlorosuccinimide and dissolved in 1.5 mL of anhydrous methylenechloride and then cooled to 0° C. under argon. To this 0.052 mL (0.71mmol, 5.1 eq.) of dimethyl sulfide was added. The white precipitate thatimmediately formed was stirred at 0° C. for 10 minutes and then at -20°C. (dry ice/ethylene glycol) for 10 minutes. To this a solution of thefreshly prepared allylic alcohol in 1.5 mL of anhydrous methylenechloride was added via cannula (the flask containing the alcoholsolution was rinsed with 0.5 mL of anhydrous methylene chloride and thisalso transferred to the reaction mixture via cannula). This was stirredat -20° C. for 15 minutes and then at 25° C. for 50 minutes. Thereaction mixture was quenched with water, diluted with methylenechloride, separated, the organic portion dried over anhydrous magnesiumsulfate, filtered, and the solvent evaporated. This was passed through acolumn of florisil with 10% ethyl acetate/hexane to give 46.7 mg (0.11mmol, 79%) of the allylic chloride. This was then dissolved in 2.0 mL ofanhydrous tetrahydrofuran in a flame-dried 50 mL round-bottomed flaskunder argon and to this a freshly prepared tetrahydrofuran solution oflithium diphenylphosphide (Ph₂ PLi, this deep orange reactant wasprepared by the quimolar addition of n-butyllithium todiphenylphosphine) was added slowly until a yellow color persisted. Thiswas then quenched with 0.5 mL of water, the tetrahydrofuran evaporated,diluted with 10 mL of methylene chloride, 6 drops of 30% hydrogenperoxide were added, and then rapidly stirred for 10 minutes. This wasdiluted with methylene chloride, dried over anhydrous magnesium sulfate,filtered, and the solvent evaporated. Purification by silica gel columnchromatography (5 to 50% ethyl acetate/hexane) afforded 29.3 mg (0.049mmol, 45%)(18% from Z-10) of the phosphine oxide 11 as a white solidafter removal from benzene, (Rf=0.3, 50% ethyl acetate/hexane), mp118°-122° C. ¹ H NMR (C₆ D₆) δ 7.83-7.78 (m, 4H), 7.05-7.03 (m, 6H),5.46 (ddt, J=14.0, 7.6, 1.2 Hz, 1H), 5.42 (d, J=2 Hz 1H), 4.99 (dd, J=2,1.2 Hz, 1H), 3.95-3.90 (m, 1H), 3.69 (dd, J=10.0, 6.4 Hz, 1H), 3.55 (dd,J=10.0, 8.8 Hz, 1H), 3.32-3.12 (m, 2H), 2.70-2.63 (m, 1H), 2.40-2.33 (m,1H), 2.26-2.19 (m, 1H), 1.94-1.87 (m, 1H), 1.83 (ddd, J=13, 7.6, 4.8 Hz,1H), 0.98 (s, 9H), 0.95 (s, 9H), 0.071 (s, 3H), 0.065 (s, 3H), 0.049 (s,3H), 0.014 (s, 3H); ¹³ C NMR (C₆ D₆) δ 145.4 (d, J=2.5 Hz), 142.0 (d,J=12.2 Hz), 132.8 (d, J=98.0 Hz) 132.7 (d, J=98.2 Hz), 131.62 (d, J=2.5Hz), 131.58 (d, J=2.6), 130.93 (d, J=9.2 Hz), 130.88 (d, J=9.2 Hz),128.42 (d, J=11.7 Hz), 128.40 (d, J=11.6), 114.0 (d, J=7.8 Hz), 112.6,67.32, 67.30, 64.1, 46.7, 44.1, 37.4, 31.2 (d, J=70.9 Hz), 25.8 (3C)25.7 (3C), 18.0 (2C), -4.8, -4.9, -5.4 (2C); IR (CHCl₃) 3020, 2956,2930, 2857, 1680, 1472, 1463, 1438, 1255, 1100 cm⁻¹ ; MS, m/z (El) 596(M⁺, 3), 540 (43), 539 (100), 407 (58), 332 (22), 202 (27), 201 (25), 75(30), 73 (86); HRMS, m/z (M⁺) calcd for C₃₄ H₅₃ O₃ Si₂ P 596.3271, found596.3277.

(-) -11a from (-)-Z-10: [α]_(D) ²³.5° C. -54.0° (c=0.061, CH₂ Cl₂, d.e.98.5%)

(+) -11a from (+)-z10: [α]_(D) ²³.5° C. -54.4° (c=0.096, CH₂ Cl₂, d.e.96.5%)

EXAMPLE 9 Iα-hydromethyl-25-hydroxyvitamin D₃ [(-)-2]

A flame-dried 10 mL round-bottomed flask was charged with 79.7 mg (0.13mmol, 1.9 eq.) of the phosphine oxide (-)-11a and dissolved in 1.0 mL offreshly distilled anhydrous tetrahydrofuran and cooled to -78° C. underargon. Phosphine oxide (-)-11a was azeotropically dried with benzene andheld under high vacuum for 24 hours immediately prior to use. To this0.091 mL (0.138 mmol, 2.0 eq.) of PhLi (1.52M in diethyl ether) wasadded dropwise over a 5 minute period. A deep orange-red color persistedafter the second drop of the PhLi solution was added. This was allowedto stir an additional 8 minutes at -78° C. at which time a precooled(-78° C.) solution consisting of 24.3 mg (0.069 mmol, 1.0 eq.) of the CDring ketone in 0.5 mL of freshly distilled anhydrous tetrahydrofuran wasadded dropwise via cannula. The C,D ring ketone 12 was alsoazeotropically dried with benzene and held under high vacuum immediatelyprior to use. The flask containing the C,D ring ketone 12 was rinsedwith 0.4 mL of tetrahydrofuran and this was also slowly added to thereaction mixture via cannula. This deep orange-red solution was stirredin the dark at -78° C. for 2.5 hours and then warmed to -65° C. over 30minutes. At this temperature, the reaction mixture turned to a lightyellow. This was immediately quenched with 0.3 mL of 2N sodium potassiumtartrate followed by the addition of dilute aqueous potassium carbonate.After warming to room temperature, the reaction was diluted withmethylene chloride, separated, the organic portion dried over anhydrousmagnesium sulfate, and filtered. Purification by silica gel columnchromatography (5% to 10% ethyl acetate/hexane) afforded 37.9 mg (0.049mmol, 69%) of the crude coupled product. This was immediately placed ina flame-dried 1O mL round-bottomed flask and dissolved in 3.0 mL offreshly distilled anhydrous tetrahydrofuran under argon. To this 0.17 mL(0.17 mmol, 3.5 eq.) of tetrabutylammonium fluoride (1M intetrahydrofuran) was added and stirred at 25° C. in the dark for 14hours. The solvent was evaporated and the crude product passed through acolumn of silica gel with 5% to 10% methanol/diethyl ether and thenpurified by PTLC (3×1000μ, 8% methanol/diethyl ether) to afford 17.2 mg(0.039 mmol, 83%) [58% from (-)-11a] of1α-hydroxymethyl-25-hydroxyvitamin D₃ [(-)-2]. This compound was onlysparingly soluble in organic solvents (e.g. MeOH, CHCl₃, CH₂ Cl₂ ). ¹ HNMR (CDCl₃) δ 6.32 (d, J=11.2 Hz, 1H), 5.95 (d, J=11.2 Hz, 1H), 5.18 (d,J=2 Hz, 1H), 5.02 (d, J=2 Hz, 1H), 0.93 (d, J=6.4 Hz, 3H), 0.54 (s, 3H).¹³ C NMR (CD₃ OD) δ 147.7, 142.6, 136.7, 124.0 119.0, 114.1, 71.5, 67.4,64.7, 58.0, 57.6, 47.4, 47.0, 46.5, 45.3, 41.9, 37.8, 37.6, 37.5, 30.0,29.3, 29.1, 28.7, 24.7, 23.3, 22.0, 19.4, 12.3; UV (EtOH) Λmax 264 nm;mp 181°-184° C.

EXAMPLE 10 1β-hydroxymethyl-3β-norhydroxy-3α,25 dihydroxyvitamin D₃[(+)-3]

This procedure was similar to the one used for the preparation ofvitamin 2. The amounts of reagents utilized were as follows:

phosphine oxide (+)-11b: 101.3 mg(0.17 mmol, 2.7 eq.),

PhLi (1.52M in Et₂ O): 0.135 mL (0.21 mmol, 3.3 eq.),

C,D ring 12: 22.3 mg (0.063 mmol, 1.0 eq.)

This afforded 21.1 mg (0.049 mmol, 76%) of the vitamin (+)-3 as an offwhite solid. ¹ H NMR (CDCl₃) δ 6.31 (d, J=11.3 Hz, 1H), 5.94 (d, J=11.3Hz, 1H), 5.15 (dd, J=2.1, 1.0 Hz, 1H), 4.99 (d, J=2 Hz, 1H), 4.03-3.97(m, 1H), 3.63-3.55 (m, 2H), 2.83-2.78 (m, 1H), 2.65-2.57 (m, 1H),2.30-2.24 (m, 1H), 0.93 (d, J=9.8 Hz, 3H), 0.5 (s, 3H); ¹³ C NMR (CDCl₃)δ 145.4, 143.3, 134.1, 123.7 117.0, 113.9, 71.1, 67.2, 64.4, 56.5, 56.3,46.3 45.9, 44.5, 40.5, 37.5, 36.4, 36.1, 29.4, 29.2, 29.1, 27.7, 23.6,22.3, 20.8. 18.8, 11.9; UV (EtOH) Λmax 264 nm; mp 118°-123° C.

The 1-hydroxymethyl derivatives of the invention have been compared withcalcitriol for biological activity. The compounds were tested for growthinhibition of murine keratinocyte cells (cell line PE) and for theinhibition of TPA-induced ornithine decarboxylase (ODC) activity.

The cell line PE was derived from a papilloma induced in female SENCARmice by a standard skin initiation/promotion protocol (Carcinogenesis,7:949-958 (1986)) and was chosen for its particular sensitivity to theinduction of ornithine decarboxylase (ODC) activity by the extensivelycharacterized tumor promoter TPA. The PE cell line culture medium usedin the tests consisted of Eagle's minimal essential medium withoutcalcium chloride supplemented with 8% chelexed fetal calf serum and 1%antibiotic-antimycotic and the addition of CaCl₂ to 0.05 mM Ca⁺⁺.

Growth Inhibition Test

The growth inhibition test was carried out as follows:

Growth curves for PE cells treated with calcitriol and its1-hydroxymethyl homologs were generated by assay for the reduction of3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)according to the method described by Carmichael et al, Cancer Res.,47:936-942 (1987). A mitochondrial dehydrogenase reduces MTT to a blueformazan product with an absorbance maximum of 505 nm in DMSO; thenumber of viable cells can thus be determined spectrophotometrically.

PE cells were seeded at a density of 5,000 cells/well in 50 μl mediuminto 96-well microtiter plates. Twelve hours later, the medium wasremoved, and cells were treated with 100 μl fresh medium into which theappropriate amount of vitamin D₃ or derivative dissolved in dimethylsulfoxide (DMSO) had been added, with the concentration of DMSO heldconstant at 0.1%. The plates were fed once at 48 hours, with thereaddition of the vitamin D₃ analogues at this time. At 24 hourintervals following the initial treatment of the cells with compounds,0.1 mg (50 μg of a 2 mg/ml solution) of MTT was added to the plates.After 4 hours, the MTT was removed and DMSo added to dissolve the blueformazan dye. Using a microtiter plate reader, the A₅₀₅ was thendetermined and cell number calculated from blank-subtracted absorbancevalues. Results from the MTT assay for the inhibition of cell growthwere independently confirmed by treating 100 cm² dishes of cells in ananalogous manner for 96 hours, whereupon the cells were harvested bytrypsinization and counted. Further, the viability of the cells treatedwith vitamin D₃ or derivatives was determined to be identical to controlcells at 96 hours by trypan blue exclusion.

Inhibition of TPA-induced ODC Activity

100 cm² dishes of PE cells were treated with vitamin D₃ or analoguesdissolved in DMSO by direct addition into the culture medium. Fifteenminutes later, the plates were treated with 100 ng/ml TPA dissolved inethanol. For both additions, the solvent concentration was held constantat 0.1%, and control values represent the results from plates treatedwith these solvents. Three plates were used for each experimental group.Following incubation for 4 hours after addition of TPA, the medium wasremoved and the dishes washed with ice cold phosphate-buffered saline(PBS). The excess PBS was then removed and the dishes rinsed with an icecold solution of pyridoxal phosphate in PBS (50 μg/ml). The excessliquid was removed, and the dishes were frozen at -80° C. The disheswere scraped into Eppendorf tubes while still partially frozen, and thecells further lysed by freeze-thawing for generation of the 12,000×gcytosol. Cytosolic ODC activity was determined in triplicate bymeasuring the release of ¹⁴ CO₂ from L-[¹⁴ C]ornithine using anEppendorf microvessel assay as previously described (Cancer Res.,43:2555-2559 (1983)).

Results

Results of the foregoing tests are illustrated by the accompanyingdrawings wherein:

FIG. I graphically shows the growth inhibition of keratinocyte cell linePE by vitamin D₃ and 1-hydroxymethyl homologs at 3 μM. The values shownrepresent the mean from 12 wells±S.D. Arrows indicate administration offresh medium into which the compounds dissolved in DMSO had been added.Control cells were treated with DMSO alone (0.1% in culture medium). Thetreated values are significantly different from the solvent control at72 and 96 hours (p 0.001, Student's t-test).

FIG. II illustrates the inhibition of TPA-induced ornithinedecarboxylase activity by pretreatment with vitamin D₃ and1-hydroxymethyl homologs. FIG. II A shows inhibition of TPA-stimulatedresponse by pretreatment of cells for 15 minutes with 1 μM of thecompounds. Values represent the mean ±S.D. for 3 measurements.Pretreatment with calcitriol or its synthetic derivatives resulted in astatistically significant reduction in TPA-induced ODC activity(p<0.001, Student's t-test). FIG. II B shows a dose-response curve forthe inhibition of TPA-induced ODC activity with the 1-hydroxymethylvitamin D₃ diastereomers (-) -2 and (+)-3.

As shown, calcitriol and its 1-hydroxymethyl derivatives were equipotentat inhibiting growth of PE cells. The antiproliferative effects of thethree compounds as demonstrated by reduction in cell number over time ascompared to control plates is shown in FIG. I. While the control cellscontinued in the exponential phase of cell growth from 24 hours onward,this rapid rate of cell proliferation was significantly blunted bytreatment with calcitriol or its 1-hydroxymethyl derivatives. Further,the treated cell populations had reached a plateau by 72 hours, daysbefore the control cells would become confluent and senescent. Thus allthree vitamin D₃ compounds were active in inhibiting cell growth anddivision. The activity of these compounds was due to cytostatic ratherthan cytotoxic effects, as cell viability was unchanged in the treatmentgroups as determined by dye exclusion assay.

Calcitriol and the 1-hydroxymethyl diastereomers also significantlyinhibited the effects of TPA (12-O-tetradecanoylphorbol-13-acetate) onthe activity of ornithine decarboxylase (ODC). ODC catalyzes the initialand rate-limiting step in the polyamine biosynthetic pathway; while thefunction of polyamines is not fully understood, they are essential forgrowth, differentiation and replication. This enzyme can be inducedrapidly and dramatically by many growth stimuli, including the tumorpromoter TPA (Annu. Rev. Biochem., 53:749-790 (1984)). The ability ofTPA to induce ODC is associated with its proliferative andtumor-promoting properties (Cancer Res., 35:2426-2433 (1975); Biochem.Biophys. Res. Commun., 105:969-976 (1982) and Proc. Natl. Acad. Sci.USA, 70:6028-6032 (1982)). A variety of agents have been shown toinhibit TPA effects on ODC induction as well as TPA-stimulated tumorpromotion, including calcitriol (Cancer Res., 45:5426-5430 (1985);Biochem. Biophys. Res. Commun., 116:605-611 (1983)) anti-inflammatorysteroids and vitamin A analogues (Biochem. Biophys. Res. Commun.,91:1488-1496 (1979)), as well as free radical scavenging compounds (Adv.Free Radical Biol. and Med., 2:347-387 (1986)). Similarly, FIG. II showsthe effects of vitamin D₃ and its 1-hydroxymethyl derivatives on theTPA-stimulated ODC activity in vitro. The potency of the three compoundsas inhibitors of the effects of TPA on this enzyme were notsignificantly different from each other. FIG. II B of FIG. IIillustrates the similar dose-response characteristics of the1-hydroxymethyl vitamin D₃ diastereomers.

Taken together, these results indicate that replacing the 1α-hydroxylgroup in calcitriol does not diminish biological activitiescharacteristic of vitamin D₃. Further, the results demonstrate thatchanging the stereochemistry of a 1-substituent (i.e., 1α->1β2->3) doesnot necessarily change antiproliferative activity.

The foregoing shows unexpectedly high antiproliferative and cell growthinhibitory activities for the 1-hydroxyalkyl analogues of the invention,the expectation from the prior art being that replacement of the1α-hydroxyl group of calcitriol would be damaging to such activities. Itis also surprising that changing the stereochemistry of the1-hydroxyalkyl (1α to 1β, compound 2 to compound 3) did not change theantiproliferative or cell growth inhibitory activity. Both (-)-2 and(+)-3 showed less than, or equal to, 2% of calcitriol's binding to the1,25(OH)₂ -vitamin D₃ receptor.

It will be appreciated that various modifications may be made in theforegoing without departing from the spirit and scope of the inventionas defined in the following claims wherein:

What is claimed is:
 1. A vitamin D₃ analogue of the formula: ##STR7##and stereoisomers thereof, wherein R is --R³ OH where R³ is straight orbranched alkyl of 1 to 6 carbons, R¹ is hydrogen and R² represents a25-hydroxy D₃ side chain.
 2. An analogue according to claim 1 which is1-hydroxymethyl-25-hydroxyvitamin D₃ of the formula: ##STR8## or theformula: ##STR9##